Chronic pain is an unsolved global healthcare problem that remains a major cause of suffering, disability, and substantial economic burden worldwide (Cohen et al., 2021). It is estimated that the rate of chronic pain in humans is as high as 20% each year (Dahlhamer et al., 2018). The International Association for the Study of Pain (IASP) defines pain as unpleasant sensory and emotional experiences related to or similar to actual or potential tissue damage (Raja et al., 20220). Pain can be categorized as acute or chronic based on the duration and nature of its occurrence. Modern medical research has proven that acute pain is a symptom that goes away as the disease gets better; However, chronic pain usually lasts for more than 1 month or even 3 months, and this pain is no longer a symptom, but should be treated as a disease. The occurrence of pain is caused by many factors. In addition to the impact of injury or disease, there are physical perception, psychological and emotional changes, genetic factors, etc. Due to the complexity of pain, the results of the current treatment of chronic pain are unsatisfactory (The, 2021). Currently available analgesics do not meet treatment goals. For example, opioids and non-steroidal anti-inflammatory drugs (nsaids), two commonly used analgesics in clinical practice, have different side effects in chronic pain. The main adverse reactions induced by opioids include analgesic tolerance, addiction and physical tolerance, nausea and vomiting, constipation, and respiratory depression (Paul et al., 2021). The long-term use of NSAIDs can produce gastric bleeding and cardiovascular-related adverse reactions (Trelle et al., 2011). The different side effects greatly limit the widespread use of these two classes of drugs. Therefore, there is an unmet need for effective analgesic drugs that are safer and more tolerable than currently available treatments.

Nerve growth factor (NGF) was first discovered by Rita Levi-Montalcini (Levi-Montalcini, 1952). NGF in the body is both an essential protein and an important pain-producing substance (Hirose et al., 2016). It has been found that NGF binding can be two types of receptors (Landreth and Shooter, 1980), the protomyosin receptor kinase a (TrkA), which has a high affinity for NGF, and the p75 neurotrophic factor receptor (p75NTR), which has a low affinity for NGF. NGF is secreted by inflammatory cells in damaged tissues and by Schwann cells in damaged nerves (Thacker et al., 2007; Basbaum et al., 2009). When tissue injury occurs, NGFs are abundantly expressed at the site of injury and bind to TrkA receptors on the cell membranes of sensory nerve endings, which in turn transmits pain signals, ultimately leading to the onset of pain. Therefore, in cases where an inflammatory response persists with damaging pain, such as low back pain, back pain, autoimmune diseases - rheumatism, etc., or in cases of neuropathic pain, NGF induces sensitization of the peripheral and central systems, which can lead to nociceptive hypersensitivity and anisocoria, and analgesics that block NGF/TrKA signaling can be very effective in these pathologies (Hirose et al., 2016). In recent years, researchers have also identified it as one of the key targets for drugs to treat chronic pain. Currently, the main NGF/TrkA inhibitor drugs are anti-NGF antibodies and TrkA activity inhibitors (Hirose et al., 2016). This study will focus on a new anti-NGF antibody, which have shown promising results in clinical studies and are expected to provide new clinical treatment strategies for chronic pain in the future.

Metabolomics is the quantitative and qualitative analysis of all small molecules in a biological sample at a certain time and under certain conditions. It is a accredited methodology with more than 20 years of practical experience. At present, metabolomics has been extended to many disciplines (Jeyarajah et al., 2006; Wishart, 2016; González-Domínguez et al., 2020). Metabolomics technologies have improved the mechanistic understanding, diagnosis, and treatment of various diseases by utilizing biological samples from humans and animal and cellular models of human diseases. Although the relationship between metabolomics and pain may be complex because the occurrence of pain is accompanied by multiple physiological symptoms, these issues have the potential to manifest through the level of metabolomics (Miettinen et al., 2021), which will facilitate the development of mechanism-based pain diagnostics to support mechanism-based therapeutic approaches. Metabolomics research has the potential to advance these goals because it can discover pain biomarkers to improve pain treatment options, facilitate drug development progress, and ultimately reduce the burden of chronic pain.

DS002 is an anti-NGF monoclonal antibody developed by Tashi Pharmaceutical (Guangdong) Co. Ltd. It interrupts the signal transmission by blocking the binding of NGF to the TrkA receptor, making it unable to stimulate the corresponding sensory neurons, and finally achieving an analgesic effect. Currently, a phase I clinical trial of DS002 has been completed and published in our laboratory (Ma et al., 2022). This study aims to explore the effects of DS002 on pain, cartilage and bone metabolism-related markers, and the changes in metabolic profile in vivo, to provide more evaluation basis for the drug development of DS002.

Pain is a common problem and is the main reason people seek medical care. But satisfactory clinical protocols for the treatment of pain remain elusive. Anti-NGF monoclonal antibody is a new class of analgesic drugs. In the studies that have been done on the treatment of pain with this class of drugs, it has shown high efficacy in the treatment of pain, far exceeding the efficacy of existing drugs, and is non-addictive. In vitro studies showed that DS002 successfully blocked the binding of NGF and TrkA receptors, thereby blocking the transmission of pain signals. DS002, as a novel analgesic drug targeting NGF, has the potential to become a new generation of drugs for treating chronic pain, nevertheless, previous studies have shown that co-targeted drugs of DS002 cause serious adverse effects on joints. In addition, the results of our Phase I clinical trial demonstrated that DS002 did not experience dose-limiting toxicity in terms of safety and did not meet dose-escalation termination criteria in the range of 0.5–20 mg administered (Ma et al., 2022). Five subjects developed arthralgia during the course of the trial, four subjects treated with DS002 (one in the 4.0 dose group, one in the 12.0 dose group, and two in the 20.0 mg dose group) and one in the placebo group, although the symptoms that developed were medically diagnosed to be mild, and all eventually resolved without intervention. No serious bone and joint events or peripheral neuropathy/sensory abnormalities were noted throughout the trial. Rapidly progressive arthritis (RPOA) was not observed even when the drug was administered at high doses. As this was the most prominent concern when the FDA shelved the development programme for similar anti-NGF drugs. Therefore, in the present study, we continued to investigate the effects of DS002 on cartilage and bone metabolism-related markers to better clarify whether it produces the same target drug-induced adverse effects in the joints and to discuss its safety in healthy subjects. Meanwhile, this study also explores the changes in metabolite levels in healthy subjects after taking DS002 from the perspective of blood metabolomics, and links the changes in metabolites to the mechanism of pain occurrence, to explore the reasons for the therapeutic effect of DS002, and to provide more scientific basis for advancing the marketing of DS002.

Previous Tanezumab clinical trial data showed joint adverse effects, including osteonecrosis and rapidly worsening osteoarthritis, in the same class as DS002 (Jayabalan and Schnitzer, 2017). Hyaluronic acid (HA) is the main component of the cartilage matrix, which can bind to CD44 receptors, thereby inhibiting the expression of interleukin (IL) 1β and reducing the production of matrix metalloproteinase 3 (MMP-3) (Karna et al., 2008). HA can enter the peripheral blood circulation through lymphatic circulation, and the expression level of HA is positively correlated with the severity of arthritis. Matrix metalloproteinases (MMPs) have been reported to play an essential role in the pathogenesis of osteoarthritis, as they are involved in the degradation of all matrices outside the cell except polysaccharides (Grillet et al., 2023; Murphy et al., 2002). Among them, MMP-3 is the most important protease involved in cartilage degradation, which can be regulated by IL-1 and TNF-α cytokines (Shi et al., 2022), followed by activation of other interstitial collagenases to degrade type II collagen (COL II), accelerates cartilage destruction. COL II is the main extracellular matrix protein in cartilage, and its degraded fragments will further accelerate the progression of osteoarthritis, promote the degradation of COL II, and accelerate the destruction of cartilage (Poole et al., 2002). Cartilage oligomeric matrix protein (COMP) is secreted by osteoblasts and synovium cells and is related to cartilage metabolism. After articular cartilage injury, cartilage matrix degradation produces COMP, which can be released into the blood (Maly et al., 2021). Bone alkaline phosphatase (BAP) is secreted by osteoblasts during the maturation stage and binds to bone matrix after being secreted to the extracellular space. It is a special sensitive index reflecting bone formation. In osteoporosis, bone calcification is insufficient. Osteoblasts proliferate actively and cannot transform into osteocytes. Osteoblasts proliferate actively and secrete a large amount of BAP into the blood. Tartrate resistant acid phosphatase 5b (TRACP-5b) is an enzyme secreted by activated bone calpain and is a good marker of bone resorption and osteoclast activity. Its concentration can be measured to understand bone metabolism status (Terpos et al., 2005). In this study, the concentrations of HA, MMP-3, COLII, COMP, BAP, TRACP-5b, which are related to cartilage and bone metabolism, did not change significantly over time in healthy subjects after treatment of different doses of DS002. These results indicated that the administration of monoclonal antibody DS002 did not cause fluctuations in the content of metabolites related to cartilage and bone metabolism, indicating that DS002 had no significant effect on the relevant parameters of cartilage and bone metabolism.

When pain occurs, a series of changes are reflected in the body’s metabolites. Specific blood, urine, and saliva biomarkers can be used to determine the degree of risk of chronic pain and can be used as prognostic markers of pain progression and treatment response (Hagedorn et al., 2021). In the present study, we measured the therapeutic effect of DS002 by investigating the blood metabolic profile. After DS002 administration, the body’s metabolism was significantly affected. The changes in metabolites involved in different metabolic pathways. According to previous studies, the metabolites in these metabolic pathways have been shown to be related to the occurrence and development of different pain. For example, researchers have found a link between amino acid metabolism and pain (Alexander et al., 2013; Teckchandani et al., 2021), the ratio of tryptophan to kynurenine is increased during back pain (Staats Pires et al., 2020), glutamate levels increase with the onset of fibromyalgia (Clos-Garcia et al., 2019), phosphocholine, alanine, and taurine are elevated in neuropathic pain (Finco et al., 2016). As the occurrence of pain is accompanied by a series of symptoms, different metabolic pathways can also indirectly affect pain. Studies have shown that the methionine metabolism pathway is related to sleep problems, and the glutathione metabolism pathway is related to obesity, and pain, sleep and obesity affect each other (Suzuki et al., 2015; Miettinen et al., 2019), so the imbalance of these two pathways will also affect pain. So we propose that the treatment of pain with DS002 may have a relationship to changes in these pathways. In our results, aromatic amino acid metabolic pathways account for the most significant proportion, such as the biosynthesis of phenylalanine, tyrosine, and tryptophan, as well as phenylalanine and tyrosine metabolism. These results may be relevant to the treatment of pain with DS002, because the amino acid metabolic pathway is related to the basal metabolic recovery of the body, and correct regulation reduces inflammatory response and affects nerve conduction. In addition, amino acids are the main substrate for producing neurotransmitters, and neurotransmitter-mediated pain regulation is the primary way.

Phenylalanine and tryptophan are essential amino acids for the human body and belong to the aromatic amino acids. Most phenylalanine in the body is oxidized to tyrosine, an amino acid associated with analgesic neurotransmitters, through the catalytic action of phenylalanine hydrogenase. The pathophysiological mechanisms underlying the onset and resolution of pain are intricate, and many different neurotransmitter systems are involved in the transmission, processing, and control of pain (Millan, 2002). In previous studies, Amino acid metabolites, including L-tryptophan, L-histidine, and L-tyrosine, were significantly decreased in the brain of pain model mice, suggesting that neuropathic pain may be promoted by the reduction of amino acids related to analgesic neurotransmitters. Therefore, we infer that the in vivo metabolic conversion of PHE to amino acids related to analgesic neurotransmitters would be involved in the therapeutic effect of DS002. Tryptophan is an essential amino acid for generating and maintaining proteins, muscles, enzymes, and neurotransmitters in the human body (Körtési et al., 2022). The tryptophan metabolism pathway includes the kynurenine, serotonin, and indole pathways. Changes in tryptophan levels can lead to imbalances in the synthesis and metabolism of downstream metabolites, which may be related to the pathological and physiological mechanisms of neurological and psychiatric diseases (Comai et al., 2020). Recent studies have shown that the dysregulation of the kynurenine pathway and the changes of metabolites in its pathway are associated with neurotoxicity and inflammation, and many metabolites are associated with chronic pain (Tanaka et al., 2021). Tryptophan enters the kynurenine metabolic pathway through indoleamine 2, 3-dioxygenase (IDO) and tryptophan 2, 3-dioxygenase (TDO) and generates kynurenine, which is irreversibly converted to kynurenic acid (KA) by kynurenine aminotransferase (KATs) (Schwarcz et al., 2012). KA is a neuroactive metabolite which belongs to the antiexcitotoxin and has neuroprotective effects (Fehér et al., 2019). The activity of KA at different targets is the primary mechanism by which it achieves specific functions. KA exerts its neuroprotective effect by antagonizing NMDA receptors (Birch et al., 1988), and previous studies have shown that marked reductions in neuroprotective metabolites occur in patients with psychiatric disorders (Athnaiel et al., 2022). KA interacts with g protein-coupled receptor 35 (GPR35) to indirectly reduce inflammation (Wirthgen et al., 2017), which is highly correlated with chronic pain. Thus, the neuroprotective and inflammatory response effects of KA suggest elevated levels of KA and other metabolites with similar properties when treating chronic pain symptoms.

In recent years, a great deal of research has been conducted on the role of 5-HT in chronic pain states.5-HT is metabolised in the body by: the small amount of tryptophan in the body is catalyzed by tryptophan hydroxylase (TPH) to generate 5-hydroxytryptophan (5-HTP). The aromatic L-amino acid decarboxylase interacts with the cofactor Pyridoxal 5-phosphate to convert 5-HTP to 5-hydroxytryptamine (5-HT) (Daubert and Condron, 2010). 5-HT, also known as serotonin, is an excitatory neurotransmitter in the human central nervous system, which causes the contraction of vascular smooth muscle, thereby potentially regulating pain (Taylor and Basbaum, 1995; Xue et al., 2023). Endogenous 5-HT was released in response to tissue injury in mast cells, platelets and endothelial cells, while multiple neuronal 5-HT receptors were detected in the periphery, suggesting that 5-HT can interfere with injurious transmission at peripheral sites (Bardin, 2011). Previous studies have also pointed to the complex relationship between 5-HT and pain, for example: intradermal administration of 5-HT to rats causes dose-dependent nociceptive hypersensitivity (Taiwo and Levine, 1992); plantar injections of 5-HT result in plantar oedema and intense flinching and licking behaviour (Sufka et al., 1992); In healthy volunteers, intradermal injections of low concentrations of 5-HT produced burning pain (Lischetzki et al., 2001), while injections into the mandibular occlusal muscle produced nociceptive hypersensitivity (Ernberg et al., 2000); Furthermore, In a carrageenan inflammatory pain model, administration of the 5-HT3 antagonist ICS-205930 by plantar administration at different times resulted in complete suppression of nociceptive hypersensitivity to foot inflammation, a process accompanied by 5-HT release (Eschalier et al., 1989). Taken together, these studies suggest that 5-HT release sensitises sensory nerve fibres in response to inflammation or tissue damage and promotes peripheral sensitisation through direct or indirect mechanisms. It has been shown that patients with chronic pain exhibit weaker endogenous pain inhibitory processes compared to pain-free individuals, and that the involvement of downward inhibition may represent an endogenous mechanism to prevent the chronicisation of pain (Ossipov et al., 2014). In vivo, the periaqueductal grey matter (PAG) is a brain region capable of activating endogenous pain inhibitory systems, and the PAG influences downstream pain modulation primarily through its interconnections with the rostral ventromedial medulla (RVM) (Ossipov et al., 2014), whereas the activation of downstream projections from the RVM induces the release of serotonin (Foo and Mason, 2003; Wei et al., 2010; Hossaini et al., 2012). 5-HT can exhibit both pro- and antinociception throughout, depending on the subtype of 5-HT receptor it activates, with activation of 5-HT1A, 5-HT1B, 5-HT1D, and 5-HT7 receptors tending to be antinociceptive, and 5-HT2A and 5-HT3 receptors tending to be proinjury sensory (Suzuki et al., 2004; Sasaki et al., 2006; Bannister et al., 2009; Dogrul et al., 2009); there is also a large body of research pointing to the inhibitory and facilitatory effects of the downstream 5-HT pathway on spinal neuronal excitability and pain-related behaviours, depending on the acute or persistent pain state (Bardin, 2011). In conclusion, these results suggest that 5-TH and the downstream 5-TH pathway play an important role in pain transmission in a complex pathological manner.

A minimal amount of tryptophan in the body can also be converted into indole and its derivatives, such as indole-3-acetic acid, indole-3-propionic acid, etc. under the action of the gut microbiota (Wei et al., 2021). Indole and its derivatives can contribute to the regulation of inflammatory responses (Venkatesh et al., 2014), which are highly correlated with the occurrence of pain. Therefore, indole and its derivatives can also affect the progression of pain. In addition, the gut microbiota is also an important regulator of visceral pain. In recent years, it has been found that the gut microbiota also plays a crucial role in chronic pain (Guo et al., 2019). In summary, these associations potentially suggest a correlation between changes in metabolite levels and the efficacy of DS002 in treating pain.

From the above, our findings suggest that DS002 has greater advantages in the treatment of pain compared with the existing drugs and the drugs in the same target class as DS002. Of course, the study has limitations. Our method of administration is a single dose in healthy people, which is different from clinical drug administration. Therefore, the application of DS002 in the clinical treatment of pain needs to be further explored.

Our results showed that DS002 caused the changes in the aromatic amino acid metabolic pathway in subjects treated with it. Among them, the changes in the concentration of L-phenylalanine, 5-hydroxytryptophan, 5-hydroxytryptamine, kynurenic acid, kynurenine, and indole-3-propionic acid may be related to the therapeutic effect of DS002 for treating pain. After administration, there were no significant changes in cartilage and bone metabolism indicators, and we preliminarily speculate that DS002 will not produce severe adverse reactions as the same target class of drugs. Our research provides a specific basis for DS002, and aromatic amino acids and their metabolites may serve as biomarkers for treating pain. Combined with the clinical results of the previous study of DS002 in our laboratory, it can more strongly demonstrate the enormous potential of anti-NGF drugs represented by DS002 in treating pain.